Chagas disease is a devastating disease of the cardiovascular and digestive systems that is caused by the protozoan parasite Trypanosoma cruzi. The disease is a major public health problem in Mexico, Central America, and South America, and ~300,000 people in the United States are estimated to be infected with the parasite, a figure that is increasing with immigration. Presently, there are no dependably effective chemotherapies for Chagas disease, and the need to validate novel drug targets and discover new drugs, particularly those that target unique parasite traits, is imperative. Our application addresses this exigency. [The overall objective of this application is to test the hypothesis that parasite and host metabolism are intimately intertwined by functionally characterizing and therapeutically validating the four] members of the equilibrative nucleoside transporter (NT) family, TcNT1, TcNT2, TcNT3, and TcNT4, that initiate the translocation of purine and pyrimidine nucleosides/nucleobases from the host into the parasite. Recently, a genome-wide RNA interference screen targeting genes in mammalian host cells authenticated purine and pyrimidine uptake from the host as critical determinants to the intracellular replicatio of T. cruzi, a process that is fundamental to disease pathogenesis. Moreover, purine scavenge by T. cruzi is known to be an indispensable nutritional function since, in contrast to the mammalian host, the parasite is auxotrophic for purines and thus, must obligatorily scavenge host purines for survival and proliferation. There are two Specific Aims. Specific Aim I will focus on the functional characterization of the four NTs. We will determine the ligand specificities and affinities for TcNT1, TcNT2, TcNT3, and TcNT4, evaluate whether each of the four NTs is expressed in mammalian forms of the parasite, and verify their subcellular locations in intact parasites. Specific Aim II affords a genetic validation of the four NTs as potential therapeutic targets. We will create null mutants deficient in TcNT1, TcNT2, TcNT3, and TcNT4 by targeted gene replacement, as well as their corresponding add-backs, assess the nutritional requirements and transport phenotypes of the null lines, test the capacity of the null mutants to infect cardiomyoblasts in vitro, and determine whether the T. cruzi knockouts exhibit a compromised virulence phenotype in Balb/c mice. Accomplishment of these experiments will verify the hypothesis that the purine and pyrimidine pathways of intracellular T. cruzi and the mammalian host are interconnected and validate these pathways, not only in the parasite but, as well, in the mammalian host, as prospective drug targets. This information will inform future drug discovery efforts to treat this devastating infection of the cardiovascular and gastrointestinal systems.